This is a proposal to renew a P41 Research Resource grant that supports the Boulder Laboratory for 3-D Electron Microscopy of Cells and Macromolecular Structures. Our goals for research in technology (R&D) include the development of methods for reliable specimen preparation, image acquisition, and image processing. In addition, we provide service, training and dissemination of software and technology to the scientific community. Our methods comprise 3-D electron microscopy aimed to resolve the structure and function of cellular organelles and macromolecular assemblies. Our facility is unique in a sense that we are working on the interface between most advanced cryo-technologies applied to frozen-hydrated specimens revealing best possible molecular resolution in 3-D, and large-scale 3-D imaging typically provided by freeze-substitution and plastic embedding of cells and tissue. This way we provide a direct link between cellular structures and macromolecular detail and atomic-scale interpretation. Freeze-substitution and plastic embedding are excellent method for high-throughput 3-D imaging of large complex systems and entire cells to a resolution of approx. 4-5 nm. This is sufficient to unambiguously detect cellular structures such as microtubules, actin filaments, mitochondria, ER systems, and even macromolecular structures such as ribosomes or other large enzyme complexes. Tomographic 3-D imaging on frozen-hydrated specimens aims for most accurate molecular detail down to 2nm resolution or even beyond. That, however, requires focusing on much smaller volumes. In this renewal we propose a series of new methods that should improve our interpretations of frozen-hydrated specimens by employing novel high-electron dense labeling techniques, specifically designed for vitrified macromolecular and cellular samples, and correlative approaches combining fluorescence light microscopy with 3-D electron microscopy. We propose new computational procedures in our software packages such as volume-averaging of 3-D data picked from tomograms and a variety of processes that will improve the resolution of tomographic 3-D reconstructions, both, during data acquisition on the microscope, and for the alignment and image correction procedures.